Characterising Cfm2, a Novel Gene Involved in Vertebrate Development

Abstract

The Cfm2 gene is conserved in vertebrates and is linked to various developmental anomalies in humans. This gene is a paralogue of another novel gene named Cfm, for caudal forebrain and midbrain. In the mouse, Cfm was found to be expressed in the neuroectoderm, which forms the future caudal forebrain and midbrain, and also in the optic rudiment, first pharyngeal arch, Rathke’s pouch, tongue muscle, lung, inner wall of the alimentary canal, genital tubercle and peripheral nerves (Hirano et al., 2005). Also in the mouse, Cfm2 was found to be expressed in the presomitic mesoderm of the segmenting somite, the optic nerve, otic capsule, peripheral nerves, tegmentum, lung and tongue muscle (Hirano et al., 2005). More recently a yeast-two-hybrid screen found that FAM101A, the human orthologue of the CFM2 protein, binds to filamin A (FLNA) (Gay et al., 2011). Most instances of the otopalatodigital (OPD) syndrome spectrum of disorders are associated with mutations in FLNA, the gene encoding FLNA, however in some instances no such mutations are found (Robertson, 2007). This has led to a hypothesis implicating CFM2, the protein product of the Cfm2 gene, in the OPD syndrome spectrum of disorders through its interactions with FLNA.

In silico analysis of the CFM and CFM2 proteins revealed this family to be very highly conserved in vertebrates with orthologues found in a number of vertebrate Classes. Orthologues were also identified in the lancelet, a primitive chordate with an ancestral relationship to the vertebrates. Phylogenetic analysis of these orthologues found the CFM and CFM2 proteins to form two distinct clades in the resulting tree, representing the CFM and CFM2 sequences respectively. These results strongly support the notion of the CFM and CFM2 sequences having arisen from a duplication event in a common ancestor of the Chordates.

Reverse transcription PCR (RT-PCR) found that the Cfm gene is expressed continuously in Xenopus laevis development, and similarly in the development of the limb in Xenopus. In situ hybridisation found this expression to be located to the lateral plate mesoderm, neural tube floor plate, hypochord and the developing proctodeum at stage 30. Cfm expression was detected in the epidermal layer of the hindlimb during limb development. RT-PCR found that Cfm2 expression is much more restricted during development. In situ hybridisation found Cfm2 expression to be localised in the developing olfactory organ, lateral line precursors, pronephros and the branchial arches at stage 40 in Xenopus development. In the limbs, in situ hybridisation revealed Cfm2 to be expressed at the joints of the bones of the limb, in a manner consistent with the sequential formation of these structures. No overlapping expression was found between these genes, consistent with the notion of independent evolution in these two genes after the aforementioned duplication event.

Down-regulation of Cfm2 using a morpholino oligonucleotide (MO) had diverse effects on Xenopus development. Cfm2 MO injected tadpoles developed at a slower rate than their wild type siblings, showed less body pigment and had a number of developmental defects. Among the developmental defects observed were craniofacial and axial malformations consistent with the phenotypes observed in the OPD syndrome spectrum of disorders. However, the most consistent phenotype seen was the irregular development of the ventral fin in Xenopus. Exogenous expression of Cfm or Cfm2 was also found to have severe effects on Xenopus development, with exogenous expression of Cfm2 found to be more severe than Cfm.

The expression patterns of Cfm2 and the developmental effects seen in Cfm2 knockdown in Xenopus appear to confirm an association between Cfm2 and the OPD syndrome spectrum of disorders. However, this association is not complete and will require further analysis and understanding of the function of Cfm2 in vertebrates. Furthermore, the results from this research have led to a proposed role for Cfm2 as an important regulator in neural crest cell migration during Xenopus laevis development.